title: Pharmacological comparison of the cloned human and rat M2 muscarinic receptor genes expressed in the murine fibroblast (B82) cell line.
abstract: The coding sequence of the human m2 receptor gene was amplified by polymerase chain reaction and stably transfected into a murine fibroblast cell line (B82). We have compared the human M2 clonal cell line (HM2-B10) with the previously established B82 cell line (M2LKB2-2) expressing the rat M2 receptor to assess drug specificity, drug selectivity and effector coupling. Both transfected cell lines showed a high level of specific, saturable [3H](-)-N-methyl-3-quinuclidinyl benzilate binding with Kd values of 243 pM (155-352 pM) and 345 pM (234-539 pM) and Bmax values of 97 +/- 4 and 338 +/- 16 fmol/10(6) cells, respectively. Inhibition of [3H](-)-N-methyl-3-quinuclidinyl benzilate binding to HM2-B10 cells and M2LKB2-2 cells showed the same rank order of potency for the antagonists: atropine > dexetimide > 4-diphenylacetoxy-N-methylpiperidine methiodide > himbacine > methoctramine > 11-[[2-[(diethylamino) methyl]-1-piperidinyl]acetyl]-5,11-dihidro-6H-pyrido-[2,3-b](1, 4)-benzodiazepine-6-one > hexahydro-sila-difenidol hydro-chloride > pirenzepine. Correlation analysis of the pKi values indicate that the expressed human and rat M2 receptors have nearly identical ligand-binding characteristics. Carbachol inhibited forskolin-stimulated cAMP formation with similar potency in both cell lines [EC50 = 2.4 microM (0.2-2.8) and 1.1 microM (0.2-5.3) for the human and rat M2 receptor, respectively]. In the M2LKB2-2 cells, carbachol slightly stimulated the [3H]inositol monophosphate formation but had no significant effect in HM2-B10 cells. In conclusion, the human and rat M2 receptors expressed in the B82 cell line have very similar binding properties but exhibit slight differences in effector coupling mechanisms.
authors.affiliation: Pharmacology, The University of Arizona, Health Sciences Center, Tucson, Arizona 85724, USA.
authors.forename: I
authors.initials: I
authors.lastname: Kovacs
authors.forename: H I
authors.initials: HI
authors.lastname: Yamamura
authors.forename: S L
authors.initials: SL
authors.lastname: Waite
authors.forename: E V
authors.initials: EV
authors.lastname: Varga
authors.forename: W R
authors.initials: WR
authors.lastname: Roeske
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D018721:Muscarinic Agonists / Q000378:metabolism; D018727:Muscarinic Antagonists / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D011976:Receptors, Muscarinic / Q000187:drug effects* / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013045:Species Specificity
chemical_list: D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D002217:Carbachol; D000242:Cyclic AMP